Laccases are a subclass of the multicopper oxidase super family of enzymes, which includes ascorbate oxidases and the mammalian protein, ceruloplasmin. Laccases are one of the oldest known enzymes and were first implicated in the oxidation of urushiol and laccol in the Oriental lacquer plant (Rhus vernicifera) by Yoshida in 1883 (Reviewed in Malmström, B. G., “Early and more recent history in the research on multi-copper oxidases” in Multi-copper oxidases, ed Messercshmidt, A. (1997), World Scientific, Singapore). Work by Bertrand in 1894-7 (Malmström, B. G) further characterized the tree laccase as well as laccases from mushrooms. Laccases are now known to be widespread in fungi (Thurston (1994) Microbiology 140:19-26) and also to occur in the entire plant family of the Anacardiaceae (Hutterman (2001) Appl. Microb. Biotechnol. 55:387-394), of which the lacquer plant is a member. There are also reports of laccase activities in a variety of other plants (Bao (1993) Science 260:672). Recently there have been several reports of bacterial enzymes that exhibit laccase activity (Diamantidis, G., et al, (2000), Soil Biology and Biochemistry, 32, 919-927; Sanchez-Amat, A., et al, (1997) A, Biochem. Biophys. Res. Commun., 240, 787-792) and genes encoding putative laccases have been identified in the genomes of many more bacteria (Alexandre, G., et al, (2000), TIBTECH, 18, 41-42; Solanon, F., et al, (2001), FEMS Microbiol. Lett., 204, 175-81).
The generally accepted reaction catalyzed by laccases is the oxidation of phenolic substrates. In the case of plant laccases this activity is believed to result in oligomerization of monolignols in the early stages of the biosynthesis of lignin (Bao 1993, supra), the most abundant aromatic polymer on earth. In contrast, fungal laccases have been implicated in the degradation of lignin—the reverse reaction—particularly by white-rot fungi (ten Have (2001) Chem. Rev. 101:3397-3413). The major target application has been in the delignification of wood fibers during the preparation of pulp.
Laccases are found in many plant pathogenic fungi and there are several reports where laccase production has been correlated with infection (Williamson (1997) Front. Sci., 199, E99-E107). However, there is little evidence of a clear direct role of the laccase in the plant pathogenesis. In the opportunistic human pathogenic fungus Cryptococcus neoformans (also Filobasidiella noeformans) there is a laccase enzyme that appears to be associated with the pathogenic phenotype. CNLAC1 is present in both pathogenic and non-pathogenic species from the genus Filobasidiella (Petter (2001) Microbiology, 147, 2029-2036.), but may play a role in protecting the pathogen from attack by the host (Liu (1999) Infect. Immun., 67, 6034-6039). There are no known such associations with bacterial laccases.
Laccases catalyze the oxidation of phenolic or other compounds with the concomitant reduction of oxygen to water (Malmström, 1997, supra). They contain four active-site copper ions that mediate electron transfer between oxidant and reductant (Thurston, 1994, supra, and Petter (2001), Microbiology, 147, 2029-2036). Although the specificity for the electron donor (substrate) is low, the specificity for the acceptor (oxygen) is absolute, see FIG. 12A. For example:4-benzenediol+O2→4-benzosemiquinone+2H2O
Substrate oxidation by the laccase is a one-electron reaction that generates a free radical from the substrate. This free radical may undergo one of several reactions: i. further enzyme oxidation to yield, for example, a quinone from phenol; ii. quenching by hydrogen abstraction; or iii. polymerization.
In special cases, oxidation of the substrate yields a stabilized radical that can abstract a hydrogen from another organic molecule, thereby returning to the ground state substrate. In this case, the initial substrate is said to act as a mediator and the final product of the reaction is the oxidized form of the second organic compound. This cycling of mediator molecules is believed to be a key element of laccase-catalyzed delignification (ten Have 2001, supra, and Leonowiccz (2001) J. Basic Microbiol. 41:185-227); see FIG. 12B.
A well-studied example of a mediator molecule is 1-hydroxy benzatriazole (HBT) (Fabbrini (2002), J. Mol. Catalysis B: Enzymatic, 16, 231-240), which forms a stable N-oxy radical species when oxidized by a laccase. The oxidized HBT is then able to react with other organic compounds by abstraction of a hydrogen and returning to the reduced state. This mediator is utilized in the oxidation of valencene to nootkatone; see FIG. 12C.
The broad substrate specificity of laccases allows their activity to be measured by the oxidation of one of several substrates, including 2,2′-azinobis(3-ethylbenzthiazoline-sulfonic acid), (ABTS), syringaldizine, and dimethoxyphenol (DMP) (Malstrom 1997, supra, Thurston 1994, supra, and Fabbrini 2002, supra,). In each case the oxidized product absorbs in the visible wavelength range and can be easily monitored in a spectrophotometer; see FIG. 12D.
The sesquiterpene nootkatone (4,4a,5,6,7,8-hexahydro-6-isopropenyl-4,4a-dimethyl-2(3II)-naphtalenone) is an important flavor constituent of grapefruit, which in isolated form is used commercially in perfumery and to flavor soft drinks and other beverages. Flavoring agents such as nootkatone are routinely used to enhance product appeal in the food and beverage industry, the cosmetic industry and the health care industry. The increased demand for flavoring agents in these industries has created a number of opportunities for biocatalysis (use of enzymes) and fermentation to compete with traditional synthetic chemistry for the production of flavors.
Current enzymatic methods for the production of nootkatone are limited in their application due to poor turnover and loss of yield at increased substrate concentrations.